Plant Physiol. (1991) 96, 597-601 Received for publication November 30, 1990 0032-0889/91/96/0597/05/$01 .00/0 Accepted February 15, 1991 Xylem Sap Proteins Charles L. Biles and Fred B. Abeles* U.S. Department of Agriculture, Agricultural Research Service, Appalachian Fruit Research Station, 45 Wiltshire Road, Kearneysville, West Virginia, 25430 ABSTRACT here suggest that some xylem proteins, such as peroxidase, in the Xylem sap from apple (Malus domestica Borkh), peach (Prunus are specifically secreted into xylem sap by cells located persica Batsch), and pear (Pyrus communis L.) twigs was col- root. lected by means of pressure extrusion. This sap contained a Peroxidase isozymes can be used to test the idea that xylem number of acidic peroxidases and other proteins. Two other sap contains a unique set of proteins. The presence of a sources of xylem sap used in this study were stem exudates and peroxidase isozyme in xylem sap which is not found in the guttation fluid. Similar peroxidases were also found in stem surrounding leaftissue can be used as evidence that specialized exudates and guttation fluids of strawberry (Fragaria x ananassa proteins are secreted into the vascular system. Since peroxi- Duch.), tomato (Lycopersicum esculentum L.), and cucumber dases catalyze polymerization reactions, experiments were (Cucumis sativus L.). lsoelectnc focusing activity gels showed conducted to test the idea that xylem sap peroxidase plays a that two peroxidases (isoelectric point [pi] 9 and pi 4.6) were role in wound plugging. present in initial stem exudates collected in the first 30 minutes after excision. Subsequent samples of stem exudate collected contained only the pi 4.6 isozyme. The pi 4.6 peroxidase isozyme MATERIALS AND METHODS was also found in root tissue and guttation fluid. These observa- tions suggest that roots produce and secrete the pi 4.6 peroxi- Plant Material dase into xylem sap. Cucumber seedlings were treated with 100 Terminal stems of apple (Malus domestica Borkh), peach microliters per liter ethylene for 16 hours and the exudate from persica and pear (Pyrus communis L.) were decapitated hypocotyl stumps was collected over a 3 hour period. (Prunus Batsch), Ethylene increased the peroxidase activity of stem exudates and collected from the field. Sections of 20 cm by approximately inhibited the amount of exudate released. These observations 1 cm were excised from 1-year-old wood. The bark from a 1- suggest that xylem sap peroxidase may play a role in plugging cm section of the lower end of the stem was ringed and damaged vascular tissue. removed. A plastic hose attached to a syringe was placed over the exposed xylem tissue. Plastic hose clamps were used to minimize leakage. Xylem sap was expressed by forcing a solution ofbromophenol blue (A600 = 0.3) through the tissue. This dye has a negative charge at pH 6. Positively charged The stem exudate of excised watermelon seedlings was dyes such as methyl green adhered to the negatively charged shown to contain numerous acidic proteins with a range of xylem. About I mL of exudate was collected by applying mol wt from 10,000 to 100,000 (4). Peroxidase, shikimate pressure to the syringe. Dilution ofxylem sap by bromophenol dehydrogenase, malate dehydrogenase, isocitrate dehydrogen- blue forcing solution was calculated by measuring the optical ase, phosphoglucoisomerase, and phosphoglucomutase were density of the solution collected at 600 nin. Using this tech- present in this exudate. In subsequent work, stem exudate nique the respective amount of forcing solution in peach, was shown to contain proteins which inhibited the growth of pear, and apple exudate was 90, 32, and 34%. The SE of these Fusarium oxysporum, the causal agent for Fusarium wilt of measurements was 5.3%. The difference in xylem sap dilution watermelon (3). The exudate which forms on the stumps of with forcing solution may be due to the difference in the size decapitated seedlings is a mixture of phloem and xylem sap. of xylem elements. Peach xylem has a larger range of cell The original purpose of this work was to see if these proteins diameters than apple and pear. This lack of uniformity would were located in either phloem or xylem sap. cause most of the forcing solution to bypass smaller cells It was initially thought that these proteins were located in because large cells are the path of least resistance. Xylem sap phloem sap. Xylem sap is thought to contain small mol wt was filtered through a 0.22 ,um nylon filter prior to concentra- inorganic ions and organic compounds and serves as a source tion on an Amicon Centricon-30 microconcentrator (W. R. of these substances and water to the leaf. However, in 1923 Grace, Danvers, MA). Wilson showed that guttation fluid (essentially xylem sap) Guttation fluid or stem exudates were collected by means from grasses contained enzymes such as catalase, peroxidase, of a handheld pipetter. Guttation fluid was collected from and reductases (10). The possibility that xylem sap contains field-grown strawberry (Fragaria x ananassa Duch.) plants. proteins raises the question oftheir source, function, and fate. Guttation fluid was also collected from greenhouse grown Are xylem sap proteins the result of breakdown products tomato plants (Lycopersicum esculentum L.). Guttation fluid produced during xylem formation or are they produced by from tomato was collected after the plants were stored in parenchyma cells adjacent to xylem tissue? The data presented 100% relative humidity chambers for 18 h. Care was taken 597 Downloaded from on January 21, 2020 - Published by www.plantphysiol.org Copyright © 1991 American Society of Plant Biologists. All rights reserved. 598 BILES AND ABELES Plant Physiol. Vol. 96, 1991 using Pharmacia gels with ampholytes in the 3 through 9 or 3 4 through 6.5 range as described previously (2). Silver staining ow was used to visualize proteins in both separation systems. The AWL. method used to silver stain gels and the mol wt and IEF "$M. -Wiobb .4.6 standards were obtained from Pharmacia. Peroxidase activity in gels was visualized with a 10 mm guaiacol/I0 mM H202 substrate. pi Spectrophotometric Analysis Protein concentration was determined by the Bio-Rad pro- tein dye assay following the directions supplied by Bio-Rad. 9 p ~ i A PR P C s T I Figure 1. Peroxidase isozymes from apple (A), pear (PR), peach (P), I* cucumber (C), strawberry (S), and tomato (T) xylem sap. Samples _. !A from apple, pear, and peach were extracted by the pressure extrusion method and concentrated 50-fold with an Amicon Centricon-30 mi- croconcentrator. The protein content of the 4 AL sample was 1.2 Mu Al for apple and 0.4 jig for pear and peach. The cucumber guttation -6. fluid was not concentrated prior to electrophoresis. The 4 ,ML sample -e contained 0.012 Mg protein. The strawberry and tomato guttation -4 fluids were concentrated 50-fold and contained 0.4 Mig protein. The arrow indicates the pi 4.6 peroxidase. -2i.:_Z4 4 . -1.} not to disrupt glandular hairs on the leaf surface. Cucumber seedlings (Cucumis sativus L. "Straight Eight") were grown T S C: P PR kt; and treated with 100 AiL/L ethylene or air for 18 h in sealed chambers (2). Guttation fluid was collected from cotyledon and leaf mar- B gins. Plants were decapitated below the cotyledon and the stem exudate collected after the cut surface was washed with water and blotted dry with a paper towel. Samples were collected every 30 min over a 3 h period. Samples were stored at -20'C until used. There were three replications per treat- ment. Each replication consisted ofthree pots with five plants per pot. Proteins were also extracted from acetone powders of roots, stems, cotyledons, and leaves of cucumber seedlings. Tissue (2 g) was homogenized in 4 mL of 70% acetone. The homog- enate was filtered through Whatman No. 1 paper on a filter funnel and washed with 100% acetone. After air drying, proteins were extracted with 1 mL of 0.1 M (pH 6.8) KPO4 in a 1.5 mL Eppendorf centrifuge tube. After centrifugation for 2 min at 15,000g, the supernatant was used in enzyme assays (1). Electrophoresis Figure 2. Xylem proteins from apple (A), pear (PR), peach (P), The SDS-PAGE was run using 10 to 15% acrylamide gradient cucumber (C), strawberry (S), and tomato (T) xylem sap. proteins used in these samples were similar to those used above except that gels from Pharmacia (Piscataway, NJ) on the Pharmacia Phastsystem. The electrophoretic and staining techniques the cucumber guttation fluid was also concentrated 50-fold and the 4 ,L sample had a protein content of 0.6 Mg protein. A, The size was run were those recommended by Pharmacia. IEF'-PAGE range of xylem sap proteins. The mol wts of the protein standards are shown in the right lane. B, The charge of these proteins. The pl Abbreviations: IEF, isoelectric focusing, pl, isoelectric point. of protein standards is indicated in the right lane. Downloaded from on January 21, 2020 - Published by www.plantphysiol.org Copyright © 1991 American Society of Plant Biologists. All rights reserved. XYLEM SAP PROTEINS 599 The specific activity of peroxidase was measured with 1 mM A guaiacol and 1 mM H202 as substrates and expressed as the 3.5 of change in absorbance at 470 nm/minm mg protein. 7-4 4.6 RESULTS IEF electrophoresis of xylem sap proteins from woody and herbaceous plants indicated the presence ofa number ofacidic peroxidases (Fig. 1). All contained a peroxidase with a pI of 4.6. Four peroxidase isozymes were evident in the xylem pi exudate of apple (A), pear (PR), and peach (P).